The introduction of protein and DNA into cells, embryos, small animals and eggs plays an important role in the fields of drug development, genetic engineering and in-vitro fertilization. It has been applied to create transgenic mammals, improve drought tolerance in plants, and to reprogram skin cells into induced pluripotent stem cells (iPS) for patent-specific stem cells. However, the efficient transfection of materials still poses a problem, and a variety of techniques, broadly classified as biological, chemical and mechanical are actively being developed.
Biological techniques employ genetically modified viruses (viral vectors) to introduce DNA into cells. This method is highly efficient, as many viruses have evolved complex mechanisms to overcome cellular barriers, but suffers from high cytotoxicity, restricted targeting of specific cell types, limited carrying capacity, production problems and high costs. Furthermore, there are concerns associated with the use of viral vectors for applications in humans, such as neutralisation by serum anti-bodies and immunogenic reactions.
This has led to the development of synthetic chemical vectors such as lipoplexes (liposome complexes) to allow delivery of material into cells. Prior to delivery, negatively charged DNA molecules are complexed with uptake enhancing transfection reagents. These complexes then bind to cells and are internalized, typically by endocytosis. Although this approach is relatively simple and safe, efficiency remains poor due to low uptake across the plasma membrane, inadequate release of DNA molecules and lack of nuclear targeting. A major barrier to efficiency is the degradation caused by enzymes within endosomes and lysosomes.
Physical methods such as the gene gun, electroporation and sonoporation overcome some of these issues by forming transient openings in the cell membrane to introduce molecules into the cytoplasm thus avoiding endosomal and possibly lysomal degradation. This is achieved either by particle bombardment or by applying electric fields or ultrasound to induce changes in membrane permeability. Although these methods increase transfection efficiency, this increase is directly related to increased cytotoxicity. Furthermore, they only provide limited, non-quantitative reagent transfection. In addition, degradation by enzymes within the cytoplasm remains a problem.
One physical method that overcomes the aforementioned problems is capillary microinjection. Transfection is achieved through the direct insertion of a microinjection needle into the cell using a microscope and precision micromanipulators. Two capillary microinjection techniques exist, differing in the mechanism used for reagent transport. These are capillary pressure microinjection (CPM), which uses pressure driven flow (PDF), and ionophoresis, which employs electrokinetic principles (electrophoresis) for reagent transport.
Of these two methods, CPM is most widely used as it is the simplest and most direct way to inject extracellular material in a targeted fashion (cytoplasm or nucleus) without cell or reagent restrictions. A wide range of substances, including naked DNA, RNA, antibodies and nanoparticles have been injected into cells with high transfection efficiency (up to 100%) and low cytotoxicity. However, the use of PDF for reagent delivery limits the efficacy of CPM. It has been shown that the volume delivered into cells may vary by a factor of five or more, resulting in significant variability and low reproducibility of injections. The use of PDF also imposes limits on the minimum injection needle size because of the high pressures that are required for dosing. Existing commercial injection systems or devices operate in the order of the 500 kPa which restricts the minimum tip diameter to 0.2-0.5 μm. This limitation on the tip diameter is significant as smaller tips lead to increased viability as they reduce the damage inflicted into cells during injection.
To enable the use of smaller needles, non-pressure driven reagent delivery such as ionophoresis may be employed. Ionophoresis involves the insertion of electrodes into the injection needle and into the cell medium to generate an electric field that induces electrophoresis. Ionophoresis overcomes the limitations on tip size as it is independent of needle geometry. However, reagent delivery is slow and is dependent on the properties of the ions to be injected. Furthermore, quantization of the reagent delivered remains a challenge. These factors make ionophoretic reagent delivery impractical for clinical studies.
In addition to the limitations associated with the reagent delivery, capillary microinjection suffers from low throughput and variability as it is an operator-mediated process. It requires a trained operator to perform the injections, which allows only for a few hundred transfections per experiment. This makes capillary microinjection impractical for studies that require statistically significant sample sizes. Furthermore, the success rate of injections is largely dependent on the operator's ability to orient the needle and holding pipette in a three-dimensional space. This results in variability and low reproducibility due to human error.
To address these issues, a number of semi and fully automated microrobotic injection systems and devices have been developed to enable rapid injection of Drosphila and Zebrafish embryos. However, these standalone systems are costly and lack integratability which makes them unsuitable for studies that require large sample sizes, as in the field of drug discovery.
This problem of integration and throughput can be addressed by the application of microfluidic technology. Performing cell injections in a microfluidic format allows for integration of pre and post-processing operations such as cell culture, sorting, and viability testing. Integration also reduces potential damage to the cell caused by changes in the cell environment. Other advantages of microfluidics include greater selectivity, reduced dead volume and automation.
Microfluidic technologies have been applied in a number of devices for microinjection of cells. Of these devices, only one approaches high-throughput injections. The device features a stationary injection needle where cells are impigned on by hydrodynamic pressure. However, the functionality of the device is limited and it is not truly scalable. As the needle is stationary in the device, it is does not compensate for differences in cell sizes and also loses location selectivity for the injection. Furthermore, the device uses pressure driven flow for injections and therefore also suffers from dosing and needle size restriction.